PROJECT SUMMARY/ABSTRACT Outer membrane vesicles (OMV) are spherical, secreted membranous structures generated from the outer membrane (OM) of Gram-negative bacteria. First discovered in the 1970's, OMV have been increasingly proposed to carry crucial roles in bacterial pathogenesis and symbiosis. Bacteroides spp. constitute up to 50% of the human gut microbiome. OMV produced by these bacteria have been assigned important roles in immunomodulation, gut inflammation, and establishment of a polysaccharide utilization network among human intestinal symbionts. For example, Bacteroides OMV are thought to prevent colitis in murine models of inflammatory bowel disease. Despite of decades of research, there is a remarkable lack of detailed molecular models to account for bacterial vesiculation, which has led to the notion that OMV could be cell lysis byproducts. In recent years, we and others have begun to identify components involved in OMV biogenesis across diverse bacterial species. Unlike OMV produced by other bacteria, Bacteroides OMV are homogenous in size and shape and do not contain any components indicative of cell lysis, such as cytoplasmic or ribosomal proteins. Instead, we have discovered that Bacteroides OMV are selectively packed with a distinct set of proteins, including multiple glycosylases and proteases that can degrade macromolecules in the gut. Importantly, these OMV proteins are hardly detectable in the OM from which they are derived. Bacteroides cell membranes contain sphingolipids and aminolipids, lipids rarely found in prokaryotes. Members of the Bacteroidetes phylum are the only gut commensals known to produce them. Recently, it has been shown that Bacteroides derived sphingolipids contribute to modulate the host immune system and maintain intestinal homeostasis. Furthermore, aminolipids help Bacteroides spp. to adapt to stress and colonize the mammalian gut. Our preliminary data demonstrate that Bacteroides OMV are enriched in certain sphingolipids and aminolipids. The goals of this proposal are a) Discriminate between ?real? OMV and lysis byproduct vesicles in cell culture; and b) Determine if particular sphingolipids and aminolipids are directed to OMV in B. thetaiotaomicron. To achieve our goals, we have identified lipoproteins differentially sorted to OMV and we will exploit this unique property of Bacteroidetes OMV to distinguish between bona-fide OMV and lysis by-products for the first time. These OMV markers will to visualize OMV formation in cell culture. Moreover, we propose to investigate the potential role of sphingolipids in OMV biogenesis. In summary, we will use biochemistry, cell biology and microscopy vo experiments in an exhaustive approach to describe OMV biogenesis down to the single-cell level. As OMV are putative large players in human gut health, with this proposal we aim to build the foundation of knowledge that will lead to novel OMV-based therapies, where engineered bacteria produce OMV with tailored cargo to modulate gut homeostasis.